What precautions can I take to ensure that my analytical and preparative gels have as similar spot patterns as possible?
To ensure preparative gels are as similar to analytical gels as possible a number of protocol variations can be tried. The sample can be processed after labelling to improve levels of protein solubilisation before IEF. For example, sonication of samples immediately prior to IEF (i.e. rehydration buffer, DTT and pharmalytes already added) can help to resolubilise proteins. Proteins can also be resolubilised after labelling by the addition of organic solvents (e.g. isopropanol) or increasing the concentration of IEF-compatible detergent (e.g. ASB14) in the sample. More consistent spot patterns can also be obtained by focusing preparative and analytical IPG strips together and using 2-D gel cast at the same time. Alternatively, a high loading of unlabelled protein can be run within a couple of analytical gels, and thus separated in the same 1st and 2nd dimension as the labelled protein. Following analysis of the CyDye DIGE Fluor labelled images, the gel is post-stained and scanned prior t
